Intestinal transport of HDND-7, a novel hesperetin derivative, in in vitro MDCK cell and in situ single-pass intestinal perfusion models.

1. Hesperetin (HDND) possesses extensive bioactivities, however, its poor solubility and low bioavailability limit its application. HDND-7, a derivative of HDND, has better solubility and high bioavailability. In this study, we investigated the intestinal absorption mechanisms of HDND-7. 2. MDCK cells were used to examine the transport mechanisms of HDND-7 in vitro, and a rat in situ intestinal perfusion model was used to characterize the absorption of HDND-7. The concentration of HDND-7 was determined by HPLC. 3. In MDCK cells, HDND-7 was effectively absorbed in a concentration-dependent manner in both directions. Moreover, HDND-7 showed pH-dependent and TEER-independent transport in both directions. The transport of HDND-7 was significantly reduced at 4 °C or in the presence of NaN3. Furthermore, the efflux of HDND-7 was apparently reduced in the presence of MRP2 inhibitors MK-571 or probenecid. However, P-gp inhibitor verapamil had no effect on the transport of HDND-7. The in situ intestinal perfusion study indicated HDND-7 was well-absorbed in four intestinal segments. Furthermore, MRP2 inhibitors may slightly increase the absorption of HDND-7 in jejunum. 4. In summary, all results indicated that HDND-7 might be absorbed mainly by passive diffusion via transcellular pathway, MRP2 but P-gp may participate in the efflux of HDND-7.

A HPLC-MS/MS method for the quantitation of free, conjugated, and total HDND-7, a novel hesperetin derivative, in rat plasma and tissues: Application to the pharmacokinetic and tissue distribution study.

A sensitive and reliable HPLC-MS/MS method was developed and validated for the determination of free (unconjugated), glucuronidated, sulfated, and total (free and conjugated) HDND-7 in rat plasma and tissues. Plasma and tissues samples were treated prior to and after the enzyme hydrolysis. Chromatographic separation was achieved on a Phenomenex Luna C18 column (150 × 4.6mm, 3 µm), using isocratic mobile phase consisting of 0.1% formic acid-acetonitrile (50:50, v/v) at a flow rate of 300 µl/min. The detection was performed on a triple quadruple tandem mass spectrometer using positive electrospray ionization (ESI) source with a chromatographic run time of 5.0 min. The detection was operated by multiple reaction monitoring (MRM) of the transitions of m/z 429.3 → 223.9 for HDND-7 and 272.9 → 152.9 for naringenin (IS), respectively. This method was validated in terms of specificity, linearity, precision, accuracy, and stability. The calibration curves for plasma and tissues were linear over a wide concentration range of 0.02-40 µg/ml with a lower limit of quantification (LLOQ) of 0.02 µg/ml. Mean extraction recoveries in plasma and tissues ranged from 87.4 to 97.1% and from 54.2 to 70.5%, respectively. The intra- and inter-day precision values were below 15% and the accuracy was within ± 15%. The samples were stable under all the tested conditions. This method has been successfully applied to the pharmacokinetic study following oral doses of 25, 50 and 100mg/kg and intravenous dose of 25mg/kg, and tissue distribution study following oral dose of 50mg/kg.

Hesperetin derivative-7 inhibits PDGF-BB-induced hepatic stellate cell activation and proliferation by targeting Wnt/ß-catenin pathway.

Liver fibrosis results from a continuous wound-healing response of the liver to repeated injury. Hesperidin (HDN) is a naturally occurring flavanone glycoside, which is extracted from fruit peels of the genus citrus. Previous studies focused on the anti-inflammation, anti-tumor and anti-oxidant roles of HDN. However, the role of HDN in hepatic fibrosis is still unknown. Here, we evaluated the role of HDND-7, a derivative of HDN which has better water solubility and bioavailability, in the activation and proliferation of PDGF-BB-induced hepatic stellate cells (HSCs), then we investigated the anti-fibrotic effect of HDND-7 in CCl4-induced mouse model of liver fibrosis. The study aimed to determine whether HDND-7 could affect the survival of HSC-T6 in vitro, while evaluating its anti-fibrotic efficacy on CCl4-induced liver fibrosis in Kunming mice. Our results revealed that HDND-7 inhibited the proliferation and activation of PDGF-BB-treated HSC-T6 cells in a time- and dose-dependent manner. In addition, administration of HDND-7 significantly attenuated liver fibrosis, as evident by the dramatic down-regulation of α-smooth muscle actin (α-SMA) and type I collagen alpha-1 (Col1α1) in both mRNA and protein levels in vivo and in vitro. Furthermore, we also found that HDND-7 decreased the expression of ß-catenin and the downstream proteins, cyclind1 and C-myc, indicating that HDND-7 may inhibit the activation and proliferation of PDGF-BB-induced HSC-T6 and attenuate liver fibrosis, at least in part, through targeting the Wnt/ß-catenin signaling pathway. Hence HDND-7 might be employed as a promising natural supplement for liver fibrosis drug therapy.